Rhabdomyosarcoma (RMS) accounts for ~8% of all pediatric cancers, where the overall 5-year survival rate for children diagnosed with metastatic RMS is less than 30%. The pro-metastatic Homeobox (Hox) transcription factor SIX1 is overexpressed in at least 10 tumor types, including RMS, where it is critical for metastasis. Six1 expression is normally limited to early embryonic development, where its promotion of precursor cell activation and migration contribute to proper formation of muscle, kidney and the inner ear. In zebrafish there are two SIX1 homologs, six1a and six1b, with little known about the role of six1a. Because developmental programs promote tumorigenesis, investigation of six1 function during embryogenesis is of great interest. Further, the mechanisms controlling six1 expression during development and tumorigenesis are yet to be elucidated. Throughout embryogenesis and myogenesis, microRNAs (miRs) have been shown to coordinate complex temporal and tissue-specific patterns of protein expression, including regulation of many Hox genes. In tumor models, miRs are shown to inhibit RMS tumor growth, and miR-mediated downregulation of Six1 can prevent kidney tumor progression. These data indicate that investigation of miRs targeting six1 in myogenesis could provide insight into new therapies for RMS. Objectives: To utilize zebrafish to assess six1a/b function and to investigate miR-mediated six1 regulation during myogenesis and tumorigenesis. Hypothesis: six1a and six1b are necessary for myogenesis, where their expression is tightly controlled by miRs 30a, 216a, and 219-3p. Aberrant expression of six1, perhaps through miR downregulation, can promote RMS onset in zebrafish. Ectopic expression of these miRs will lead to six1 downregulation and inhibition of RMS progression. Specific aims: 1) To investigate the six1a/b functions during zebrafish muscle development. 2) To determine whether miRs 30a, 219-3p and/or 216a regulate six1a/b during zebrafish myogenesis. 3) To investigate the roles of six1a/b and miRs 30a, 216a and/or 219-3p in RMS initiation and progression. Morpholino-mediated knockdown and mRNA/miR overexpression through injection into 1-4 cell embryos will be utilized to manipulate in vivo gene expression in zebrafish, followed by in situ hybridization, real-time PCR mRNA quantification, immunoblot and immunofluorescent protein detection, and other morphologic analyses to assess phenotypes. Potential roles for six1a/b and regulatory miRs will be analyzed in an established zebrafish RMS model, and further explored through model development by driving six1a/b overexpression in rag2-positive muscle progenitors. SIX1 and miR expression in human RMS tumors will be analyzed to assess disease stage correlation. Many developmental genes promote cell properties, such as survival and migration. These genes are downregulated during development, but are often re-activated in cancer cells. Research into how six1a and six1b are controlled during both embryogenesis and tumorigenesis can provide insight into therapeutic interventions, not only for RMS patients, but for those afflicted with any of the numerous cancers where SIX1 is active.